Establishment Of Rapid Detection Methods For Shellfish Viral Pathogens OsHV-1 And HaHV-1

In recent years,Chin’ese aquaculture industry has been developed rapidly.Intensive farming patterns and frequent trade flows have led to more aquatic epidemic diseases,seriously affecting the green and healthy development of aqu aculture industry and causing huge economic losses to the country and farmers.Of those,45 billion was lost in 2018,with shellfish accounting for about 20%.In 2019,the Ministry of Agriculture and Rural Affairs,among others,asked all localities to strengthen prevention and control of aquatic animal diseases.However,shellfish lack effective pathogen detection technology,which restricts the effective prevention and control of relative epidemic diseases.In this paper,the methods of modern biological techniques for detection of aquatic pathogens,including nucleic acid molecular biology,immunology and cell culture,are reviewed.For shellfish virus detection,ordinary PCR or fluorescence quantitative PCR test are commonly used,which requires professional knowledge of operat ors and highly depends on instrument.In addition,the detection method has the drawbacks of tedious nucleic acid extraction and high false positive rate.Ther efore,a rapid and accurate method for rapid detection of shellfish pathogens is urgently needed.Oyster herpes virus（Ostreid herpesvirus 1,Os HV-1）,a diseased shellfish that develops gill erosion and blood lymphocytes seep out until the individual dies.Haliotid herpes virus 1（Ha HV-1）,with its short incubation period,acute onset,high infectivity and high mortality.For these two major shellfish viral diseases,a rapid method for detection of shellfish viral diseases at protein and nucleic acid levels has been established for the first time.The main findings of this paper are as the follows:1.Rapid detection of shellfish viral pathogens at protein levels1)Development of a rapid method for detection of Os HV-1 based on immunochromatography assayFirstly,according to the prediction of protein structure and previous research reports,oSHV-1 membrane molecular proteins ORF25 and ORF72 were screened and primers containing polyclonal sites were designed as antigen molecules to be detected.The polyclonal antibody pAb of OSHV-1 membrane molecular protein ORF25 and ORF72 protein was prepared by recombinant expression and purification in vitro,and was labeled with colloidal gold.At the same time,the bottom plate（support plate）,detection film（sandwich method with sheep anti-rabbit IgG antibody printed quality control line and pAb printed detection line）,colloidal gold marker pad,sample pad and water absorption pad were prepared,and tested successively after assembly.This study developed a colloidal gold dipstick for rapid detection of oyster herpes virus OSHV-1,which has simple operation,low cost and can realize rapid detection on site,greatly improving the detection efficiency and accuracy of such pathogens,and better serving the pathogen monitoring in the shellfish industry.2.Rapid detection of shellfish viral pathogens at nucleic acid level:First,a cohort of six primers（Suite1-Suite6）were designed according to the conservative regions of seven oSHV-1 variants:KP412538.1、GQ153938.1、NC＿005881.2、MG561751.2、KY242785.1、KY271630.1 and MF509813.1.Osh v-1 DNA samples were diluted 10 times gradient（108-101）and used as amplified DNA template.Six sets of primers were subjected to nucleic acid constant temperature amplification reaction（37℃,20min）.After 20 times of DILUTION of RPA amplification products,the samples were analyzed by using colloid gold lateral flow immunochromatographic strip（TS101,Gen Dx）.The results showed that among the six designed primers,Suite5 primer combination had higher detection sensitivity than other primer combinations,with up to 10 copies.At the same time,specificity detection was carried out.The positive DNA samples of oyster,scallop,tegillarca granosa and cocklesca granosa and healthy DNA samples were subjected to nucleic acid constant temperature amplification reaction using Suite5 primer combination.The results showed that Suite5 primers could specifically detect o SHV-1 positive samples of four bivalves,and there was no amplification band in the detection line of OSHV-1 negative samples2)Establishment of Recombinant Enzyme Polymerase Isothermal Amplifica tion（RPA）for Rapid Detection of Shellfish Virus Ha HV-1First,a total of five primers（Suite1-Suite5）were designed based on the conserved regions of two Ha HV-1 mutant strains,KU096999.1 and JX453331.1in Gen Bank,and a 10-fold gradient dilution(10^8-10¹)of Ha HV-1 DNA samples was used as an amplified DNA template for nucleic acid amplification of six primers（37°C,20 min）.After dilution of 20-fold RPA amplification products,the samples were analyzed by colloidal gold lateral flow immunochromatograp hy（TS101,Gen Dx）.The results showed that among the five designed primers,Suite5 primer combination had higher detection sensitivity than other primer combinations,and the detection sensitivity could reach 10²copies.At the same time,specificity detection was carried out,and nucleic acid isothermal amplification reaction was performed on HAHV-1 positive DNA samples and healthy a balone DNA samples using Suite5 primer combination.The results showed that Suite5 primer combination could specifically detect ha HV-1 positive samples,and there was no amplification band in the detection line of HAHV-1 negative samples.Taken together,the colloidal gold test strips developed in this study for rapid detection of the oyster herpes virus OSHV-1 are simple to use,do not require accompanying reagents and equipment,are easy to carry and preserve,have a short detection time,and allow for rapid field testing,but still have limitations of batch-to-batch antibody potency and specificity instability.A universal RPA nucleic acid isothermal amplification primer and its detection method for bivalve shellfish infected with Os HV-1 and a universal RPA nucleic acid isothermal amplification primer and test kit were also developed.It greatly satisfies the needs of frontline farmers and import and export banks for rapid on-site detection of shellfish pathogens,and also provides new ideas for detection of other aquatic animal pathogens and a good technical guarantee for rapid on-site detection of aquatic pathogens.